Unveiling the structural adaptation of a robust thermostable paraoxonase from Bacillus sp. strain S3wahi: insights...

A thermostable paraoxonase (S3wahi-PON) from Bacillus sp. strain S3wahi was recently characterised and shown to possess stability across a broad temperature range. This study expands upon the initial biochemical characterisation of S3wahi-PON by investigating the structural determinants and conformational adaptability that contribute to its thermostability, using an integrated approach that combines biophysical techniques and molecular dynamics (MD) simulations across a temperature range of 10 °C to 90 °C. Biophysical analyses confirmed that S3wahi-PON retains broad stability between 10 °C and 60 °C, with its highest structural compactness and integrity observed at 30 °C - an unusual profile compared to most thermostable enzymes, which typically peak near their upper thermal tolerance. MD simulations revealed that S3wahi-PON maintains its globular stability via a synergistic interaction between α-helical content and intramolecular forces such as hydrogen bonding, salt bridges, and hydrophobic clusters. Notably, an inverse relationship between the radius of gyration (Rg) and solvent-accessible surface area (SASA) was observed at 50 °C and 60 °C, suggesting internal tightening of the structure without a corresponding increase in surface exposure, which appears to be a promising mechanism for preserving thermostability. Moreover, loop 16, encompassing Pro192 and located near the catalytic site, exhibited pronounced flexibility that was suggested to influence the enzyme's catalytic performance. These findings indicate that the thermostability of S3wahi-PON is not governed by a single dominant feature but rather by the cooperative contribution of multiple structural elements, which collectively preserve its catalytic conformation under thermal stress. Overall, S3wahi-PON emerges as a promising moderately thermostable enzyme suitable for the bioremediation of organophosphate (OP)-contaminated water systems. The insights gained from this study advance our understanding of its stability mechanisms and provide a foundation for future protein engineering strategies to enhance its applicability in diverse environmental and industrial contexts.